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Zhang H.,CAS Wuhan Botanical Garden | Zhang H.,University of Chinese Academy of Sciences | He D.,CAS Wuhan Botanical Garden | Yu J.,Asia Pacific Application Support Center | And 5 more authors.
Proteomics | Year: 2016

Seed germination is an important aspect of the plant life cycle, during which, reactive oxygen species (ROS) accumulate. The accumulation of ROS results in an increase in protein oxidation of which carbonylation is the most canonical one. However, there is insufficient information concerning protein oxidation, especially carbonylation and its contribution to seed germination. In this study, biotin hydrazide labeled chromatography combined with sequential window acquisition of all theoretical fragment ion spectra (SWATH) method was used to analyze the dynamic pattern of protein carbonylation in rice embryos during germination. A total of 1872 unique proteins were quantified, among which 288 carbonylated peptides corresponding to 144 proteins were determined based on the filtering through mass shifts of modified amino acids. In addition, 66 carbonylated proteins were further analyzed based on their carbonylation intensity in four stages of germination. These identified carbonylated proteins were mainly involved in maintaining the levels of ROS, abscisic acid and seed reserves. Remarkably, a peroxiredoxin was found with 23 unique carbonylated peptides, and the expression of which was consistent with its increased activity. This study describes the dynamic pattern of carbonylated proteins during seed germination, and may help to further understand the biochemical mechanisms on this process. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

Du Z.,Asia Pacific Application Support Center
Chinese Journal of Chromatography (Se Pu) | Year: 2013

A new method has been developed using a hybrid triple-iadrupole linear ion trap (QTrap) mass spectrometer for the fast detection and identification of nine β-agonists, clen-buterol, salbutamol, ractopamine, ritodrine, terbutaline, isoxsuprine, tulobuterol, cimaterol and bambuterol, in one single liquid chromatograpny-tandem mass spectrometry (LC-MS/MS) analysis. The homogenized tissue samples were purified with liquid-liquid extraction after enzy-matic hydrolysis by β-glucuronidase/aryl snlfatase. After gradient elution separation on Cl8 LC column using acetonitrile and formic acid aqueous solution as the mobile phases, a multiple reaction monitoring (MRM) scan as survey scan and an enhanced product ion (EPI) scan as dependent scan were performed in an information dependent acquisition (IDA) experiment. Finally, the identification of the drugs was carried out by library search with a newly developed MS/MS library based on EPI spectra at three different collision energies in positive mode. The analytical method in the present study was well validated and good results were obtained with respect to precision, repeatability and spiked recovery. The limits of detection of residues were 0. 1-0. 2 μkg for β-agonists, and with a linear range from 0. 1 to 50. 0 μg/L. Three concentration levels of 0. 5, 1. 0 and 5. 0 μg/kg were spiked in pig tissues, and the overall recoveries were between 72. 0% and 95. 1% with the relative standard deviations (RSDs) between 3. 1% and 12. 1%. The real sample test showed that this method could be used for sensitive and accurate determination of β-agonist residues in pig tissues. Source

Du Z.,Asia Pacific Application Support Center
Chinese Journal of Chromatography (Se Pu) | Year: 2011

A method was developed for the simultaneous determination and identification of 12 steroid hormone residues in pig tissues, including stanolone, aldosterone, boldenone, danazol, metandienone, methyltestosterone, nadrolone, norethindrone, progesterone, stanozolol, testosterone and testosterone propionate, using liquid chromatography-tandem triple-quadrupole linear ion trap mass spectrometry(LC-MS/MS). Homogenized pig tissue samples were purified with a Waters MCX solid phase extraction column after enzymatic hydrolysis by β-glucuroni-dase, then separated on a Venusil MP C 18 column (100 mm x 2. 1 mm, 3 μm) using gradient elution with the mobile phases of acetonitrile and water with 0. 1% (v/v) formic acid. A multiple reaction monitoring (MRM) as survey scan and an enhanced product ion (EPI) scan as dependent scan were performed in an information dependent acquisition (IDA) experiment. The compound identification was carried out by library search with a newly developed MS/MS library based on EPI spectra at three different collision energies in positive mode. The results showed that the limits of detection (LODs, S/N = 3) were in the range of 0. 2 - 0. 5 μg/kg for the steroid hormones; and with a good linearity (r >0. 99) ranged from 0. 5 to 100. 0 μ/L. The average recoveries (n =6/ of the 12 steroid hormones spiked in pig tissue samples at 5. 0 μ/kg ranged from 72. 0% to 98. 1% with the relative standard deviations (RSDs) between 3. 1% and 12. 5%. The method was applied for the qualitative and quantitative determination of steroid hormone residues in pig tissues with sensitive and accurate characteristics. Source

Liu X.,Jilin University | Hu L.,Jilin University | Ge G.,CAS Dalian Institute of Chemical Physics | Yang B.,Jilin University | And 5 more authors.
Proteomics | Year: 2014

Cytochrome P450 (CYP) is one of the most important drug-metabolizing enzyme families, which participates in the biotransformation of many endogenous and exogenous compounds. Quantitative analysis of CYP expression levels is important when studying the efficacy of new drug molecules and assessing drug-drug interactions in drug development. At present, chemical probe-based assay is the most widely used approach for the evaluation of CYP activity although there are cross-reactions between the isoforms with high sequence homologies. Therefore, quantification of each isozyme is highly desired in regard to meeting the ever-increasing requirements for carrying out pharmacokinetics and personalized medicine in the academic, pharmaceutical, and clinical setting. Herein, an absolute quantification method was employed for the analysis of the seven isoforms CYP1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1 using a proteome-derived approach in combination with stable isotope dilution assay. The average absolute amount measured from twelve human liver microsomes samples were 39.3, 4.3, 54.0, 4.6, 10.3, 3.0, and 9.3 (pmol/mg protein) for 1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1, respectively. Importantly, the expression level of CYP3A4 showed high correlation (r = 0.943, p < 0.0001) with the functional activity, which was measured using bufalin-a highly selective chemical probe we have developed. The combination of MRM identification and analysis of the functional activity, as in the case of CYP3A4, provides a protocol which can be extended to other functional enzyme studies with wide application in pharmaceutical research. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

Wu X.,Xiamen University | Tian L.,Xiamen University | Li J.,Xiamen University | Zhang Y.,Xiamen University | And 10 more authors.
Molecular and Cellular Proteomics | Year: 2012

Receptor interacting protein 3 (RIP3) is a protein kinase that plays a key role in programmed necrosis. Despite the importance of RIP3-dependent necrosis in many pathological processes, current knowledge on the function of RIP3 is very limited. Here we present the results of a proteome-wide analysis of RIP3-regulated phosphorylation sites using cells from wildtype (RIP3 +/+) and RIP3 knockout (RIP3-/-) mice. Because the activation of RIP3 requires stimulation by certain extracellular stimuli such as ligands of death receptors or Toll-like receptors, we compared the phosphorylation sites of lipopolysaccharide (LPS)-treated peritoneal macrophages from RIP3+/+and RIP3-/- mice and the phosphorylation sites of tumor necrosis factor (TNF)-treated RIP3+/+ and RIP3 -/- mouse embryonic fibroblast (MEF) cells. Stable isotope labeling by amino acids in cell culture and spike-in stable isotope labeling by amino acids in cell culture were used in the analyses of the MEFs and macrophages, respectively. Proteomic analyses using stable isotope labeling by amino acids in cell culture coupled with immobilized metal affinity chromatography-hydrophilic interaction liquid chromatography fractionation and nanoLC MS/MS identified 14,057 phosphopeptides in 4306 proteins from the macrophages and 4732 phosphopeptides in 1785 proteins from the MEFs. Analysis of amino acid sequence motifs among the phosphopeptides identified a potential motif of RIP3 phosphorylation. Among the phosphopeptides identified, 73 were found exclusively in RIP3+/+macrophages, 121 were detected exclusively from RIP3 +/+ MEFs, 286 phosphopeptides were induced more in RIP3+/+ macrophages than in RIP3-/- macrophages and 26 phosphopeptides had higher induction in RIP3+/+MEFs than in RIP3-/- cells. Many of the RIP3 regulated phosphoproteins from the macrophages and MEF cells are functionally associated with the cell cycle; the rest, however, appear to have diverse functions in that a number of metabolism related proteins were phosphorylated in macrophages and development related phosphoproteins were induced in MEFs. The results of our phosphoproteomic analysis suggest that RIP3 might function beyond necrosis and that cell type specific function of RIP3 exists. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Source

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